Rabbit anti-rat polyclonal anti-GABA_B1 R (cat. no. 3835; dilution, 1:1,000), rabbit anti-rat polyclonal anti-GABA_B2 R (cat. no. 3839; dilution, 1:1,000), rabbit anti-rat polyclonal anti-β-actin (cat. no. 4967; dilution, 1:1,000), and goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (cat. no. 7074; dilution, 1:1,000) were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). All other chemicals used in the present study, unless otherwise stated, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

GLGZD consists of six drugs: Trichosanthes kirilowii Maxim, Paeonia lactiflora Pall, Cinnamomum cassia Presl, Glycyrrhiza uralensis Fisch, Zingiber officinale Rosc and Ziziphus jujuba Mill, in a weight ratio of 10:3:3:2:3:3. Dried crude drugs were purchased from Guo Yi Tang Chinese Herbal Medicine store (Fujian, China), and the drug mixture was soaked in double distilled water for 30 min. Subsequently, the mixture was decocted twice by boiling (1 h each time). The filtered solution from the two decoctions was then concentrated using a rotary evaporator (Model RE-2000; Yarong Biochemistry Instrument Factory, Shanghai, China), in order to obtain a final concentration of 1.06 g/ml. The GLGZD was subsequently stored for further analysis.

Male Sprague-Dawley rats (n=27; initial body weight, 240–280 g) were purchased from Shanghai Laboratory Animal Center (Shanghai, China), and housed in a temperature- and humidity-controlled room. The rats underwent a 12 h light/dark cycle and were provided access to food and water ad libitum. All animal treatments and experiments were approved by the Institutional Animal Care and Use Committee of Fujian University of Traditional Chinese Medicine (Fuzhou, China).

The cerebral I/R model was established following middle cerebral artery occlusion (MCAO), as described previously (28). Briefly, prior to surgery the rats were fasted for 24 h; during this time the rats were still provided ad libitum access to water. Following anesthesia with 10% chloral hydrate by intraperitoneal injection (300 mg/kg), the left common carotid artery, left external carotid artery (ECA), and internal carotid artery (ICA) were exposed and isolated by a midline neck incision. Nylon surgical thread (18–22 mm) was inserted into the left ICA until the blunted distal end met resistance, in order to block the middle cerebral artery (MCA). A total of 2 h after occlusion, reperfusion was achieved by removing the thread to restore blood supply to the MCA. The rectal temperature of the rats was maintained at 37°C throughout the surgical procedure.

The rats were randomly divided into the following three groups: i) Sham-operated control (SC) group (n=9), which underwent neck dissection and the exposure of ICA and ECA, without MCAO; ii) ischemia control (IC) group (n=9), which underwent MCAO surgery followed by reperfusion 2 h after occlusion; and iii) GLGZD group (n=9), which underwent MCAO surgery followed by reperfusion 2 h after occlusion, and received GLGZD (14.3 g/kg body weight) by intragastric administration once a day for a period of 7 days, as soon as the rats had recovered from reperfusion.

A total of 2 h after induction of cerebral ischemia, and for the following 7 days, neurological defects of the rats were scored every other day. The neurological defects were scored by two researchers, who were blinded to the treatment conditions, according to a standard scoring system on a four-point scale (28): Score 0, no neurological defect; score 1, unable to fully extend the right forepaw; score 2, circling to the right; score 3, falling to the right; and score 4, loss of walking ability. The rats that scored between 1 and 3 points were considered to be successful models.

In order to measure the muscle tone, strength, stamina and balance of the rats subjected to cerebral ischemia, screen tests were performed 2 h after induction of cerebral ischemia and for the following 7 days, using a net screen (Dashitong Equipment Co., Ltd., Fuzhou, China). The screen tests were conducted as described previously (29). The scoring criteria were as follows: 5, Holding onto the screen and climbing upward; 4, holding onto the screen with forelimbs and not falling down within 5 sec; 3, holding onto the screen temporarily and slipping off a certain distance; 2, falling to the ground within 5 sec; 1, falling to the ground immediately, as soon as the screen was set at a vertical position.

A total of 7 days after induction of cerebral ischemia, the rats were sacrificed with 10% chloral hydrate, and perfused transcardially with 0.9% NaCl. The brain of each rat was harvested and dissected into six coronal blocks (2 mm/slice). The fresh slices were incubated in 2% (w/v) 2,3,5-triphenyltetrazolium chloride solution in phosphate-buffered saline for 20 min at 37°C in the dark. Normal areas of the brain were stained dark red based on intact mitochondrial function, whereas the infarct areas remained unstained. Images of the stained slices were captured using a high-resolution digital camera (Canon Sx20; Canon, Inc., Tokyo, Japan). The Motic Med 6.0 Digital Medical Image Analysis system (Motic Microscopes, Xiamen, China) was used to quantify the infarct volume by integration of the areas from the sections.

Total RNA was extracted from the cortical infarct region of the rats using TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions. Purified RNA (3 µg) was reverse transcribed into cDNA using the RevertAid™ H Minus First Strand cDNA Synthesis kit (Fermentas, Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's instructions. The obtained cDNA was used to determine the mRNA expression levels of GABA_B1 R and GABA_B2 R by PCR using Taq DNA polymerase (Fermentas); β-actin served as an internal control. An S1000 PCR thermal cycler (Bio-rad Laboratories, Inc., Hercules, CA, USA) was used and the PCR conditions were as follows: 94°C for 3 min, denaturation for 30 sec at 94°C (35 cycles), annealing for 30 sec at 58°C, polymerization for 45 sec at 72°C and finally extension for 10 min at 72°C. The primers and the annealing temperatures (°C) used for the amplification were as follows: Sense, 5′-CGG GTG GTA TGC TGA CAA CTG GT-3′ (23 bp, 58°C) and antisense, 5′-ATG TTG GAA ATG CTT CGG GTG TT-3′ (23 bp, 58°C) for GABA_B1 R; sense, 5′-GGA CTT CAA CTA CAC AGA CCA CAC GC-3′ (26 bp, 58°C) and antisense, 5′-TTG TAT TCG CCG ACC TTC ACC TCT CT-3′ (26 bp, 58°C) for GABA_B2 R; and sense, 5′-CTA TCG GCA ATG AGC GGT TC-3′ (20 bp, 58°C) and antisense, 5′-ACT GTG TTG GCA TAG AGG TCT-3′ (21 bp, 58°C) for β-actin. Samples were analyzed by 1.5% agarose gel electrophoresis (Biowest, Hong Kong, China). The DNA bands were then examined using a Gel Documentation system (Gel Doc 2000; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The cortical infarct region of the rats was homogenized in non-denaturing lysis buffer (EMD Millipore, Boston, MA, USA) and centrifuged at 12,000 × g for 10 min. The protein concentration in the supernatants was then determined. Protein lysates were separated by 10% SDS-PAGE and then electrophoretically transferred to polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology, Shanghai, China). The membranes were blocked for 2 h with 5% non-fat dry milk, and were then incubated with primary antibodies targeting GABA_B1 R, GABA_B2 R and β-actin (1:1,000 dilution) overnight at 4°C. The membranes were then incubated with appropriate HRP-conjugated secondary antibodies for 1 h. The blots were visualized using enhanced chemiluminescence, and images were captured using a Bio-Image Analysis system (Bio-Rad Laboratories, Inc.).

All data are presented as the means of ≥3 determinations. Statistical analysis of the data was performed with Student's t-test and one-way analysis of variance using the SPSS package for Windows version 16.0 (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The neuroprotective effects of GLGZD were examined by evaluating the neurological defect scores and cerebral infarct volume of the rats. As shown in Fig. 1, all of the rats in the IC and GLGZD groups exhibited marked signs of cerebral infarction and neurological defects, whereas the rats in the SC group did not show any manifestation of cerebral injury. However, following treatment with GLGZD for 7 days, the cerebral infarct volumes were significantly reduced (Fig. 1A and B), and the neurological defect scores were improved (Fig. 1C). These results indicate that GLGZD may have therapeutic efficacy against cerebral I/R injury.

To determine the motor function of the rats subjected to cerebral I/R, screen tests were performed on the rats from all of the experimental groups. As shown in Fig. 2, the screen test scores of the IC rats were significantly decreased, as compared with the rats in the SC group. However, disordered motor function was significantly ameliorated following treatment with GLGZD between days 5 and 7. These results indicate that treatment with GLGZD may ameliorate cerebral I/R-induced spasticity in rats.

To further explore the mechanisms underlying the GLGZD-induced amelioration of post-cerebral I/R spasm, RT-PCR and western blot analysis were performed to detect the mRNA and protein expression levels of GABA_B R subunits in the ischemic cortex, respectively. The mRNA and protein expression levels of the GABA_B1 R and GABA_B2 R were decreased in the IC group, as compared with the SC group. However, following treatment with GLGZD for 7 days, the expression levels of the GABA_B1 R and GABA_B2 R were increased (Fig. 3 and 4). These data suggest that treatment with GLGZD may promote the activation of the GABA_B R in the ischemic cortex.